Purifying insulin using cation exchange and reverse phase chromatography in the presence of an organic modifier and elevated temperature

ABSTRACT

A process is described for purifying insulin and insulin analogs that comprises high-pressure liquid chromatography with an acidic cation exchange medium performed in the presence of a water miscible organic modifier and at an elevated temperature followed by reverse phase chromatography performed in the presence of a water miscible organic modifier and at an elevated temperature.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/124,080 filedSep. 7, 2016,as a National Stage of International Application No.PCT/US2015/019864 filed on Mar. 11, 2015, which claims the benefit under35 U.S.C. 119(e) of U.S. Provisional Application No. 61/953,001, filedMar. 14, 2014, both of which are incorporated herein by reference intheir entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “23742-US-CNT-SEQTXT-23AUG2019.txt”, creation date of Aug. 23,2019, and a size of 3 KB. This sequence listing submitted via EFS-Web ispart of the specification and is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention relates to a process for purifying insulin andinsulin analogs that comprises high-pressure liquid chromatography withan acidic cation exchange medium performed in the presence of a watermiscible organic modifier and at an elevated temperature followed byreverse phase chromatography performed in the presence of a watermiscible organic modifier and at an elevated temperature.

(2) Description of Related Art

Recombinant production of insulin and insulin analogs in geneticallymodified microbial or yeast host cells entails expression of the insulinor insulin analog in the host cell as a single-chain precursor insulinmolecule comprising three polypeptide domains (A-chain, C-chain,B-chain), as in native pro-insulins, along with the addition of a fusionpeptide at the N-terminus whose function is to protect the nascentprotein from internal degradation/modification during synthesis in thebacterial host, improve protein expression in the host, and to add atrypsin cleavage site that will render the correct amino acid,phenylalanine, at position B1, post-digest

In the microbial host E. coli, the single-chain precursor insulinmolecule is sequestered in inclusion bodies consisting of mostly ofincorrectly folded single-chain precursor molecules. To producerecombinant insulin, the inclusion bodies are extracted from the cell,washed, and the single-chain precursor molecule is solubilized,refolded, and then purified by at least one chromatographic step. Therefolded, single-chain precursor molecule is then further processed intoinsulin by the concurrent removal of the C-chain and the N-terminalfusion peptide by enzymatic digestion. Insulin is comprised of anA-chain and B-chain linked together by three disulfide bridges. Insubsequent purification steps, including ion-exchange chromatography andreverse phase HPLC, insulin is purified away from digestion byproductsto yield a highly purified product. The purified product may beformulated in a zinc and m-cresol (preservative) containing buffer toprovide the insulin drug product.

Methods for isolating inclusion bodies, refolding and enzymaticallydigesting precursor insulin molecules to produce insulin have beendisclosed U.S. Pat. Nos. 5,663,291; 5,986,048; 6,380,355, and 5,473,049.Ion-exchange chromatography methods for purifying insulin from digestionbyproducts have been disclosed in U.S. Pat. No. 5,101,013, whichdiscloses cation-exchange chromatography on strongly acidic ionexchangers under atmospheric or medium pressure and elution by means ofaqueous alkanol with only a relatively small amount of alkanol to purifyinsulin; and, U.S. Pat. No. 5,977,297, which discloses high-pressurecation-exchange chromatography on pressure-stable acidic cationexchangers under a pressure of about 1.1 MPa (11 bar) to about 40 MPa(400 bar) to purify insulin. Further purification of insulin has beendescribed in U.S. Pat. Nos. 6,710,167 and 5,621,073.

While there are methods available for purifying insulin, there remains aneed for alternative methods for purifying insulin and insulin analogsthereof.

BRIEF SUMMARY OF THE INVENTION

In an effort to provide additional processes for obtaining properlyfolded insulin or insulin analog from enzymatic cleavage reactions inhigh yield and pharmaceutically acceptable purity, we have found thatinsulin and insulin analogs may be purified from impurities and otherbyproducts of the enzymatic digestion by chromatography of a mixturecomprising the insulin or insulin analog on an acid-stable cationexchange chromatography in the presence of a water miscible organicmodifier and at an elevated temperature followed by reverse phase highperformance liquid chromatography in the presence of a water miscibleorganic modifier and at an elevated temperature. In particularembodiments of the process, both chromatography steps are performed atan acidic pH. The two-step chromatographic process yields apharmaceutically pure composition of the insulin or insulin analog. Theprocess has a combined yield of about 65% or greater and provides acomposition of the isolated insulin or insulin analog having a purity ofabout 99% or greater.

Therefore, the present invention provides a process for isolatingproperly folded insulin or insulin analogs from an aqueous mixturecomprising the insulin or insulin analog and related impurities, whereinthe process comprises (a) performing an acid-stable cation exchangechromatography with the aqueous mixture in the presence of a first watermiscible organic modifier and at an elevated temperature to yield afirst insulin or insulin analog mixture; and (b) performing a reversephase high performance liquid chromatography on the first insulin orinsulin analog mixture in the presence of a second water miscibleorganic modifier and at an elevated temperature to provide a secondmixture comprising the properly folded insulin or insulin analog.

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the second mixture is obtainedfrom the reverse phase high performance liquid chromatography using alinear gradient comprising the second water miscible organic modifierincreasing in concentration from about 13 to 15 percent by volume toabout 25 to 27 percent by volume. In a further aspect of the process,the linear gradient is performed on a column over a course of about 6column volumes and at about a 10 minute residence time. In a furtheraspect of the process, the process is performed on a column and thelinear gradient is performed at a rate of change of about 1.7 percent to2.3 percent of water miscible organic modifier per column volume(Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 2.0 percent of water miscible organic modifier percolumn volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa. Inparticular aspects of the process, the acid-stable cation exchangechromatography has an outlet temperature greater than room temperature;greater than 30° C.; greater than 40° C.; or a temperature between 41°C. to 45° C.; or about 42° C.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length. Inparticular aspects of the process, the reverse phase high performanceliquid chromatography has an outlet temperature greater than roomtemperature; greater than 30° C.; greater than 40° C.; or a temperaturebetween 40° C. to 46° C.; or about 43° C.

The present invention further provides a process for purifying aproperly folded insulin or insulin analog from a mixture comprising theinsulin or insulin analog and related impurities, the processcomprising: (a) applying the mixture to an acid-stable cation exchangechromatography matrix; (b) washing the matrix with a first wash solutioncomprising a concentration of mineral salt of about 10 to 25 mM and afirst water miscible organic modifier and then washing the matrix with asecond wash solution comprising a concentration of mineral salt greaterthan the concentration of mineral salt in the first wash solution andless than the concentration of mineral salt capable of eluting theinsulin or insulin analog from the matrix and the first water miscibleorganic modifier; (c) eluting the insulin or insulin analog from thematrix with an eluting solution comprising the concentration of mineralsalt capable of eluting the insulin or insulin analog from the matrixand the first water miscible organic modifier to provide a secondmixture; and (d) applying the second mixture to a reverse phase highperformance liquid chromatography matrix and eluting the insulin orinsulin analog with a linear gradient of a second water miscible organicmodifier of increasing concentration from about 13 to 15 percent byvolume to about 25 to 27 percent by volume to provide a mixture of theproperly folded insulin or insulin analog; wherein the acid-stablecation exchange chromatography is at an outlet temperature greater thanroom temperature; greater than 30° C.; greater than 40° C.; or atemperature between 41° C. to 45° C.; or about 42° C. and the reversephase high performance liquid chromatography is performed at an outlettemperature greater than room temperature; greater than 30° C.; greaterthan 40° C.; or a temperature between 40° C. to 46° C.; or about 43° C.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step: (d) applying the secondmixture to a reverse phase high performance liquid chromatographymatrix; washing the matrix with a first solution comprising about 4 to 7percent of a second water miscible organic modifier; washing the matrixwith a linear gradient of the second water miscible organic modifier ofincreasing concentration from about 4 to 7 percent by volume to about 13to 15 percent by volume; and eluting the insulin or insulin analog witha linear gradient of a second water miscible organic modifier ofincreasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedinsulin or insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl.

In a further aspect of the process, the mixture that is loaded onto thecation exchange chromatography matrix comprises from about 3.0 to about26.0 g of protein per liter in a solution comprising a water miscibleorganic modifier. In a further aspect, the mixture comprises from 10 toabout 50 percent by volume of a water miscible organic modifier. In afurther aspect, the loading step is performed using an aqueous mixturewhich comprises from about 25 to about 35 percent or 30 percent byvolume of a water miscible organic modifier. In particular aspects, thewater miscible organic modifier is hexylene glycol.

In a further aspect of the process, the elution of the insulin orinsulin analog is isocratic.

The present invention further provides a process for purifying aproperly folded insulin or insulin analog from a mixture comprising theinsulin or insulin analog and related impurities, the processcomprising:

-   -   (a) a cation exchange chromatography step performed in a column        under a column differential pressure of less than 1.1 MPa, or        less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa and        at an outlet temperature greater than room temperature; greater        than 30° C.; greater than 40° C.; or a temperature between        41° C. to 45° C.; or about 42° C., the steps comprising        -   (i) applying the mixture to a cation exchange chromatography            matrix in the column;        -   (ii) washing the matrix with a first wash solution            comprising a concentration of mineral salt of about 10 to 25            mM and a first water miscible organic modifier and then            washing the column with a second wash solution comprising a            concentration of mineral salt greater than the concentration            of mineral salt in the first wash solution and less than the            concentration of mineral salt capable of eluting the insulin            or insulin analog from the column and the first water            miscible organic modifier;        -   (iii) eluting the insulin analog from the matrix with an            eluting solution comprising the concentration of mineral            salt capable of eluting the insulin or insulin analog from            the column and the first water miscible organic modifier to            provide a second mixture; and    -   (b) a reverse phase high performance liquid chromatography being        performed at an outlet temperature greater than room        temperature; greater than 30° C.; greater than 40° C.; or a        temperature between 40° C. to 46° C.; or about 43° C., the steps        comprising        -   (i) applying the second mixture to a reverse phase high            performance liquid chromatography matrix and        -   (ii) eluting the insulin or insulin analog with a linear            gradient of a second water miscible organic modifier of            increasing concentration from about 13 to 15 percent by            volume to about 25 to 27 percent by volume to provide a            mixture of the properly folded insulin or insulin analog.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step (b) a reverse phase highperformance liquid chromatography being performed at an outlettemperature greater than room temperature; greater than 30° C.; greaterthan 40° C.; or a temperature between 40° C. to 46° C.; or about 43° C.,the steps comprising

-   -   (i) applying the second mixture to a reverse phase high        performance liquid chromatography matrix;    -   (ii) washing the matrix with a first solution comprising about 4        to 7 percent of a second water miscible organic modifier;    -   (iii) washing the matrix with a linear gradient of the second        water miscible organic modifier of increasing concentration from        about 4 to 7 percent by volume to about 13 to 15 percent by        volume; and    -   (iv) eluting the insulin or insulin analog with a linear        gradient of a second water miscible organic modifier of        increasing concentration from about 13 to 15 percent by volume        to about 25 to 27 percent by volume to provide a mixture of the        properly folded insulin or insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl. In particular aspects, the column is washed withabout 5 to 20 volumes of the wash solution.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl. In a further aspect, the column iswashed with about 15 to 25 volumes of the second wash solution.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl. In particular aspects, the insulin or insulinanalog is eluted from the column with about 20 column volumes of theeluting solution.

In a further aspect of the process, the mixture that is loaded onto thecation exchange chromatography matrix comprises from about 3.0 to about26.0 g of protein per liter in a solution comprising a water miscibleorganic modifier. In a further aspect, the mixture comprises from 10 toabout 50 percent by volume of a water miscible organic modifier. In afurther aspect, the loading step is performed using an aqueous mixturewhich comprises from about 25 to about 35 percent or 30 percent byvolume of a water miscible organic modifier. In particular aspects, thewater miscible organic modifier is hexylene glycol.

In a further aspect of the process, the elution of the insulin orinsulin analog is isocratic.

In a further aspect of the process, the differential pressure is lessthan 1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276MPa.

In further aspects of the process, the insulin is native human, porcine,or bovine insulin. In further aspects, the insulin analog is anacid-stable insulin analog, which is stable and soluble in acidic orweakly acidic solutions and insoluble or partially insoluble atphysiological pH, or a pI-shifted insulin analog in which the pI of theinsulin analog is less than or greater than the pI of native humaninsulin. The pI of native insulin is 5.4 to 5.6 thus a pI shiftedinsulin analog has a pI greater than 5.6 or less than 5.4. In particularaspects the insulin analog has a pI from between 5.8 to 8.0. Anacid-stable insulin analog such as insulin glargine has a pI of about6.7 to 7.0. In a further aspect, the insulin analog is insulin glargine,insulin aspart, insulin glulisine, or insulin lispro. For chromatographyof acid-stable insulins a weak cation material may be used and forchromatography for insulins with a pI similar to that of native insulin,a strong cation exchange material may be used.

In general, for insulin analogs with a pI greater than the pI of nativeinsulin, the cation exchanger is a weak cation exchange and for insulinanalogs with a pI similar to that of native insulin, the cationexchanger is a strong cation exchanger.

Definitions

As used herein, the term “insulin” means the active principle of thepancreas that affects the metabolism of carbohydrates in the animal bodyand which is of value in the treatment of diabetes mellitus. The termincludes synthetic and biotechnologically derived products that are thesame as, or similar to, naturally occurring insulins in structure, use,and intended effect and are of value in the treatment of diabetesmellitus.

The term “insulin” or “insulin molecule” is a generic term thatdesignates the 51 amino acid heterodimer comprising the A-chain peptidehaving the amino acid sequence shown in SEQ ID NO: 1 and the B-chainpeptide having the amino acid sequence shown in SEQ ID NO: 2, whereinthe cysteine residues a positions 6 and 11 of the A chain are linked ina disulfide bond, the cysteine residues at position 7 of the A chain andposition 7 of the B chain are linked in a disulfide bond, and thecysteine residues at position 20 of the A chain and 19 of the B chainare linked in a disulfide bond.

The term “insulin analog” as used herein includes any heterodimer analogthat comprises one or more modification(s) of the native A-chain peptideand/or B-chain peptide. Modifications include but are not limited tosubstituting an amino acid for the native amino acid at a positionselected from A4, A5, A8, A9, A10, A12, A13, A14, A15, A16, A17, A18,A19, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B15, B16, B17, B18,B20, B21, B22, B23, B26, B27, B28, B29, and B30; and/or deleting any orall of positions B1-4 and B26-30. Insulin analogs include moleculeshaving one to 10 amino acids at the N or C terminus of the A-chainpeptide and/or B-chain peptide. Insulin analogs further includemolecules amidated at the C-terminus of the A-chain peptide and/orB-chain peptide. Examples of insulin analogs include but are not limitedto the insulin analogs disclosed in published international applicationWO20100080606, WO2009/099763, and WO2010080609, the disclosures of whichare incorporated herein by reference. Insulin glargine (Gly(A21),Arg(B31), Arg(B32)-human insulin: A-chain peptide SEQ ID NO:3; B-chainpeptide SEQ ID NO:4), insulin lispro (Lys(B28), Pro(B29)-human insulin:A-chain peptide SEQ ID NO:1; B-chain peptide SEQ ID NO:5, insulinglusiline (Lys(B3), Glu(B29)-human insulin: A-chain peptide SEQ ID NO:1;B-chain peptide SEQ ID NO:6), and insulin detemir (Lys-myristicacid(B29)-human insulin: A-chain peptide SEQ ID NO:1; B-chain peptideSEQ ID NO:2 with B-29 acylated with myristic acid) are examples ofcommercially available insulin analogs.

The term “insulin analogs” further includes heterodimer polypeptidemolecules that have little or no detectable activity at the insulinreceptor but which have been modified to include one or more amino acidmodifications or substitutions to have an activity at the insulinreceptor that has at least 1%, 10%, 50%, 75%, or 90% of the activity atthe insulin receptor as compared to native insulin. In particularaspects, the insulin analog is a partial agonist that has from 2× to100× less activity at the insulin receptor as does native insulin. Inother aspects, the insulin analog has enhanced activity at the insulinreceptor.

The term “properly folded” refers to insulin or insulin analogs in whichthe cysteine residues a positions 6 and 11 of the A chain are linked ina disulfide bond, the cysteine residues at position 7 of the A chain andposition 7 of the B chain are linked in a disulfide bond, and thecysteine residues at position 20 of the A chain and 19 of the B chainare linked in a disulfide bond.

The term “pharmaceutically pure” refers to an insulin or insulin analogthat is greater than 99 percent pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a CEX chromatography gradient profile. Following loading ofthe insulin sample from an enzymatic digest in buffer A1, the column iswashed with A1 buffer (Wash 1) for 20 column volumes. Next, the columnis washed with Wash 2 containing an A1/B1 mixture (about 6.5% B1) for 20column volumes as shown. The insulin sample is eluted with an A1/B1mixture (about 26% B1) for twenty column volumes as shown. The column isstripped using B1 buffer.

FIG. 2 shows a reverse phase high performance liquid chromatographyprofile. Following loading of the insulin sample from CEXchromatography, the column is washed with Wash 1 containing about 5%water miscible organic modifier. Next, the column is washed with Wash 2containing a linear gradient of the water miscible organic modifier from5% to 14%. The insulin sample is eluted with a linear gradient of thewater miscible organic modifier from 14% to 26%.

DETAILED DESCRIPTION OF THE INVENTION

Precursor insulin or insulin analog molecules produced in prokaryotehost cells such as E. coli or lower eukaryote host cells such asSaccharomyces cerevisiae or Pichia pastoris are enzymatically cleaved invitro to remove the connecting peptide joining the B-chain peptide tothe A-chain peptide to produce insulin or an insulin analog. Theenzymatic cleavage of precursor insulin or insulin analog molecules isusing achieved by digestion with trypsin, carboxypeptidase, lysC, orcombinations thereof. However, the enzymatic digests introduceimpurities such as miscleaved protein, the three amino acid B-chaintruncate (des-Thr), deamidoinsulin, arginine- and diarginine-insulin andinsulin ethyl ester. In addition, precursor insulin or insulin analogmolecules produced in prokaryote host cells are not properly folded,therefore, the precursor molecules are subjected to a refold reactionprior to the enzymatic digest to refold the molecules into the propertertiary conformation. However, a portion of the molecules from therefold reaction are misfolded and need to be removed. In addition, somemolecules may include amino acid misincorporations and need to beremoved as well.

To remove these impurities, the present invention provides a two-stepchromatography process that is capable of providing pharmaceuticallypure compositions of the insulin or insulin analog has been developed.In the first step, a mixture comprising the insulin or insulin analogmolecules is subjected to acid-stable cation exchange chromatography inthe presence of a water miscible organic modifier and at an elevatedtemperature. The insulin or insulin analog recovered from the first stepis subjected to reverse phase high performance liquid chromatography inthe presence of a water miscible organic modifier and at an elevatedtemperature. The two-step chromatographic process yields apharmaceutically pure composition of the insulin or insulin analog. Theprocess has a combined yield of about 65% or greater and provides acomposition of the isolated insulin or insulin analog having a purity ofabout 99% or greater.

Cation Exchange Chromatography

The cation exchange chromatography process may be performed in a columnchromatography format in which during the chromatography the columndifferential pressure is less than 1.1 MPa and the outlet temperature isat an outlet temperature greater than room temperature; greater than 30°C.; greater than 40° C.; or a temperature between 41° C. to 45° C.; orabout 42° C. In particular aspects, the differential pressure is lessthan 1.0 MPa. In a particular aspect, the differential pressure may beabout 1.1 MPa to about 40 MPa as disclosed in U.S. Pat. No. 5,977,297.

The acidic cation exchange material may be a temperature-stable acidcation exchange material, which in a further still aspect, may be ahigh-capacity hydrogel polymerized within the pores of a rigid ceramicbead and cross-linked to sulfo groups on the beads, for example,BioSepra CM Ceramic HyperD F ceramic beads comprising a highlysubstituted hydrogel comprising carboxymethyl functional groups therein(available from Pall Corporation, Port Washington, N.Y.) is a weakcation exchanger whereas BioSepra S Ceramic HyperD F ceramic beadscomprising a highly substituted hydrogel comprising sulfonic acidfunctional groups therein (available from Pall Corporation, PortWashington, N.Y.) is a strong cation exchanger. In other aspects, thetemperature-stable acidic cation exchange material may be a copolymer ofpolystyrene and divinylbenzene, which has been modified with sulfogroups. Thus, acidic cation exchange materials that may be used in theprocess include but are not limited to BioSepra CM Ceramic HyperD Fceramic beads comprising a highly substituted hydrogel therein, whichare available from Pall Corporation, Port Washington, N.Y.; BioSepra CMCeramic HyperD F ceramic beads comprising a highly substituted hydrogeltherein, which are available from Pall Corporation, Port Washington,N.Y.; Source 30S or 15S polystyrene/divinyl benzene polymer resins,which are available from GE Healthcare Life Sciences, Pittsburgh, Pa.;POROS 50 μm resins, which are available from Life Technologies,Carlsbad, Calif.), and MACROPREP 50 μm methacrylate resins, which areavailable from by Biorad Corp. (Hercules, Calif.).

The acidic cation exchange material may be packed into a column forchromatography using known, conventional methods. In general, the acidiccation exchange material is equilibrated in a strip/storage buffersolution and then packed into a column. After the column is packed withthe acidic cation exchange material, the column is briefly flushed witha cleaning solution comprising a base, for example NaOH, as a pre-usesanitization step and then promptly flushed with a pre-equilibrationsolution followed by the strip/storage solution. The acidic cationexchange material should be exposed to the cleaning solution and thepre-equilibration solution for as little time as possible to reducedownstream pressure issues from occurring during the purification ofinsulin or insulin analog.

In general, the eluents (strip/storage solution, pre-equilibrationsolution, and elution solution) comprise a buffer substance, water, andorganic modifiers. Suitable buffer substances include phosphates, alkalimetal or alkaline earth metal salts, such as potassium acetate, ammoniumcitrate, sodium citrate, acetate, sulfate or chloride.

The eluents further contain water-miscible organic modifiers such asalcohols, ketones, methyl acetate, dioxane, or acetonitrile. Alcoholssuch as hexylene glycol, n- or iso-propanol, methanol, ethanol, orbutanol may be used as the water-miscible organic modifier. Theconcentration of the water-miscible organic modifier for thechromatography is from about 10 to about 50% by volume, from about 20 toabout 40% by volume, or from about 25 to about 35% by volume. Theconcentration of the buffer substance is from about 1 mM to about 100mM, about 10 to 50 mM, about 50 mM, or about 20 mM. Further additivesinclude a physiologically tolerated mineral salt such as NaCl and mayinclude one or more organic acids such as formic acid, acetic acid,lactic acid or citric acid, a base, e.g., NaOH, and/or preservatives.The pH of the buffer solution comprising the buffer substance is fromabout 2.5 to about 5.5. In particular aspects, the pH is about 5.1.

In general, the pre-equilibration solution will comprise a buffersolution; the equilibration solution will comprise a buffer solution, awater-miscible organic modifier, and a mineral salt, for example NaCl;the strip/storage solution will comprise a buffer solution, awater-miscible organic modifier, and an amount of mineral salt; forexample NaCl greater than the amount in the equilibration solution andof a concentration sufficient to remove any proteins or impurities boundto the acidic cation exchange material in the column, e.g., about 10 to25 times, about 12.5 times the amount of mineral salt in theequilibration solution.

Loading the column, chromatography, and elution of the insulin orinsulin analog is achieved using known, conventional technical methods.The loading of the column with a loading solution comprising the insulinor insulin analog to be purified may have a protein content of about 3.0to 26.0 grams of insulin or insulin analog per liter of acidic cationexchange material. In general, the loading of the acidic cation exchangematerial may be achieved by dissolving the insulin or insulin analogmixture in a buffer solution similar to the equilibration buffer for theacidic cation exchange material as described herein to provide a samplesolution. In particular aspects, the insulin or insulin analog isprovided in a sample solution with a pH of about 3.5 to 5.1, or about4.2, at a concentration of about 1 to 2 g/L, which may further include awater miscible organic modifier. In particular aspects, the samplesolution comprises from 10 to about 50 percent by volume of a solutioncomprising a water miscible organic modifier. In a further aspect, theloading step is performed using an aqueous mixture which comprises fromabout 25 to about 35 percent by volume of a solution comprising a watermiscible organic modifier. In particular aspects, the water miscibleorganic modifier is hexylene glycol.

Following the loading of the insulin or insulin analog onto the column,the column is washed in a step-wise fashion with a first wash solutioncomprising a concentration of mineral salt of about 10 to 20 mM for atleast four or five column volumes up to about 20 column volumes. Thefirst wash may be followed by an optional step of washing the columnwith a second wash solution comprising a mineral salt at a concentrationthat is greater than the mineral salt concentration in the first washsolution and less than a mineral salt concentration capable of elutingthe insulin or insulin analog from the column prior to eluting theinsulin or insulin analog for at least 15 to 20 column volumes. Elutionof the insulin or insulin analog is achieved by applying to the columnan elution solution comprising a concentration of mineral salt capableof eluting the insulin or insulin analog from the column for at least 10column volumes or for a time until the amount of insulin or insulinanalog detected by UV monitoring in a fraction collected during theelution is about 10% of the peak fraction of insulin or insulin analogduring the elution as determined by UV monitoring. Yield may bemonitored by measuring UV absorbance of fractions collected during theelution and purity may be determined by HPLC. The mineral salt may beNaCl.

In a further aspect, following the loading of the insulin or insulinanalog onto the column, the column is washed in a step-wise fashion witha first wash solution comprising about 18 to 22 mM of a mineral salt forat least five to 10 column volumes, which may be followed by an optionalsecond wash with a second wash solution comprising about 35 to 39 mM ofa mineral salt for at least 10 column volumes. Elution of the insulin orinsulin analog is achieved by applying to the column an elution solutioncomprising about 80 to 100 mM of a mineral salt for at least 10 columnvolumes or for a time until the amount of insulin or insulin analogdetected by UV monitoring in a fraction during the elution is about 10%of the peak fraction of insulin or insulin analog during the elution asdetermined by UV monitoring. Yield may be monitored by measuring UVabsorbance of fractions collected during the elution and purity may bedetermined by HPLC. The mineral salt may be NaCl.

In a further aspect, following the loading of the insulin or insulinanalog onto the column, the column is washed in a step-wise fashion witha first wash solution comprising about 18 to 22 mM of a mineral salt forabout 18 to 20 column volumes, which may followed by an optional secondwash with a second wash solution comprising about 35 to 39 mM of amineral salt for about 18 to 20 column volumes. Elution of the insulinor insulin analog is achieved by applying to the column an elutionsolution comprising about 80 to 100 mM of a mineral salt for 18 to 20column volumes or for a time until the amount of insulin or insulinanalog detected by UV monitoring in a fraction during the elution isabout 10% of the peak fraction of insulin or insulin analog during theelution as determined by UV monitoring. Yield may be monitored bymeasuring UV absorbance of fractions collected during the elution andpurity may be determined by HPLC. The mineral salt may be NaCl.

In a further aspect, following the loading of the insulin or insulinanalog onto the column, the column is washed in a step-wise fashion witha first wash solution comprising about 18 to 22 mM of a mineral salt forat least five to 10 column volumes, which may be followed by an optionalsecond wash with a second wash solution comprising about 35 to 39 mM ofa mineral salt for at least 10 column volumes. Elution of the insulin orinsulin analog is achieved by applying to the column an elution solutioncomprising about 93 to 97 mM of a mineral salt for at least 10 columnvolumes or for a time until the amount of insulin or insulin analogdetected by UV monitoring in a fraction during the elution is about 10%of the peak fraction of insulin or insulin analog during the elution asdetermined by UV monitoring. Yield may be monitored by measuring UVabsorbance of fractions collected during the elution and purity may bedetermined by HPLC. The mineral salt may be NaCl.

In a further aspect, following the loading of the insulin or insulinanalog onto the column, the column is washed in a step-wise fashion witha first wash solution comprising about 18 to 22 mM of a mineral salt forabout 18 to 20 column volumes, which may followed by an optional secondwash with a second wash solution comprising about 35 to 39 mM of amineral salt for about 18 to 20 column volumes. Elution of the insulinor insulin analog is achieved by applying to the column an elutionsolution comprising about 93 to 97 mM of a mineral salt for 18 to 20column volumes or for a time until the amount of insulin or insulinanalog detected by UV monitoring in a fraction during the elution isabout 10% of the peak fraction of insulin or insulin analog during theelution as determined by UV monitoring. Yield may be monitored bymeasuring UV absorbance of fractions collected during the elution andpurity may be determined by HPLC. The mineral salt may be NaCl.

In a further aspect, following the loading of the insulin or insulinanalog onto the column, the column is washed in a step-wise fashion witha first wash solution comprising 20 mM±2.5 mM of a mineral salt forabout 18 to 20 column volumes, which may optionally be followed by asecond wash with a second wash solution comprising 37 mM±2.5 mM of amineral salt for about 18 to 20 column volumes. Elution of the insulinor insulin analog is achieved by applying to the column an elutionsolution comprising 95 mM±2.5 mM of a mineral salt for 18 to 20 columnvolumes or for a time until the amount of insulin or insulin analogdetected by UV monitoring in a fraction during the elution is about 10%of the peak fraction of insulin or insulin analog during the elution asdetermined by UV monitoring. Yield may be monitored by measuring UVabsorbance of fractions collected during the elution and purity may bedetermined by HPLC. The mineral salt may be NaCl.

The temperature during the chromatography and elution is greater than20° C. or 30° C. or greater than 40° C. In particular embodiments, thetemperature during the chromatography and elution may be about 25° C. toabout 50° C. or from about 37° C. to about 47° C. In particularembodiments, the temperature during the chromatography and elution isgreater than greater than 40° C. In particular aspects, the temperatureduring the chromatography and elution is about 42° C. or more or about42° C. to about 47° C. In particular aspects, the temperature during thechromatography and elution is about 42° C. or at least 42° C.Preferably, the temperature is kept constant or nearly constant over thecourse of the chromatography and elution. The operating pressure duringthe chromatography and elution is substantially constant. Thechromatography and elution may be carried out using a columndifferential pressure that is less than 1.1 MPa, less than 1.0 MPa, lessthan 0.5 MPa, or about 0.276 MPa. The eluent flow rates may be fromabout 182 to about 201 cm/hour.

In another aspect, the elution may be performed with a mineral saltgradient over time, for example, by a low mineral salt concentrationbeing present in the elution buffer at the start of the elution (whichinitially can be zero percent salt) and by increasing the mineral saltconcentration during the elution process. In particular aspects, themineral salt may be NaCl. The operating temperature and pressure duringthe chromatography is substantially constant. The chromatography may becarried out using a column differential pressure that is less than 1.1MPa, less than 1.0 MPa, less than 0.5 MPa, or about 0.276 MPa and at atemperature of about 25° to about 50° C., or of about 38° to about 50°C., or of about or at least 42° C. or more, or of about 42° to about 47°C., or of about 42° C., or at least 42° C. Yield may be monitored bymeasuring UV absorbance and purity may be determined by HPLC. Inparticular aspects, the first wash solution, the optional second washsolution, and the elution solution, each comprises from 10 to about 50percent by volume of a water miscible organic modifier. In a furtheraspect, the loading step is performed using an aqueous mixture whichcomprises from about 25 to about 35 percent by volume of a watermiscible organic modifier. In particular aspects, the water miscibleorganic modifier is hexylene glycol.

In further aspects of the process disclosed herein, the first and secondwash steps and the eluting steps are performed using a first, second,and eluting solution, each comprising an aqueous mixture having about 25to about 35 percent by volume of a water miscible organic modifier,which in a further aspect, the first and second wash steps and theeluting step are performed using an aqueous mixture which comprises fromabout 30 percent by volume of a water miscible organic modifier. In afurther aspect of the process, the water miscible organic modifier ishexylene glycol.

Reverse Phase High Performance Liquid Chromatography

The reverse phase high performance liquid chromatography process may beperformed in a column chromatography format.

The reverse phase high performance liquid chromatography may beperformed with a temperature stable and pressure stable organic modifiedchromatography material or matrix. The material may be a lipophilicallymodified silica gel to which a hydrophobic matrix has been applied.Examples of a hydrophobic matrix are alkanes with a chain length of from3 to 20 carbon atoms, in particular 8 to 18 carbon atoms. Additionally,the particle size can vary within a wide range, for example from 5 to300 μm or 5 to 60 μm, in particular from 10 to 50 μm. The pore width canalso vary within a wide range; favorable pore widths are from 50 to 300Å, in particular 100 to 200 Å. Examples of lipophilically modifiedsilica gel materials are: NUCLEOSIL, Macherey & Nagel GmbH+Co.KG, Duren,Germany spherical and non-spherical materials of various particle sizeup to 45 μm, 100 Å pore width, C8- or C18-modified; LICHROPREP, E. MerckCo., Darmstadt, Germany non-spherical and spherical materials of variousparticle sizes up to μm, 60-250 Å pore width, C8- or C18-modified;LICHROSPHER SELECT B, E. Merck Co., Darmstadt, Germany sphericalmaterial up to 25 μm particle size, C8-modified; WATERS PREP, MilliporeGmbH, Eschborn, Germany C18-modified, 50-105 μm non-spherical, 100 Åpore width; ZORBAX PRO10, DuPont de Nemours (Germany) GmbH, Bad Homburg,Germany C8-modified, 10 μm, spherical, 100 Å pore width; and KROMASIL,EKA Nobel Co., Nobel Industries, Sweden C4-, C8- and C18-modified, up to20 μm, spherical, 100, 150 or 200 Å pore width. In particular aspects,the chromatography material is a silica-based reverse phase resinmodified with hydrocarbon chains about 8 carbons in length. In a furtheraspect, the chromatography material is KROMASIL C8-modified.

The reverse phase material may be packed into a column forchromatography using known, conventional methods. In general, thereverse phase material is equilibrated in a strip/storage buffersolution and then packed into a column. After the column is packed withthe reverse phase material, the column is briefly flushed with acleaning solution comprising a base, for example NaOH, as a pre-usesanitization step and then promptly flushed with a pre-equilibrationsolution followed by the strip/storage solution.

In general, the eluents (strip/storage solution, pre-equilibrationsolution, and elution solution) comprise a buffer substance, water, andwater miscible organic modifier. Suitable buffer substances includephosphates, alkali metal or alkaline earth metal salts, such aspotassium acetate, ammonium acetate, sodium citrate, acetate, sulfate orchloride.

The eluents further contain a water-miscible organic modifier such asalcohol, ketone, methyl acetate, dioxane, or acetonitrile. Alcohols suchas hexylene glycol, n- or iso-propanol, methanol, ethanol, or butanolmay be used as the water miscible organic modifier. The concentration ofthe buffer substance is from about 90 mM to about 110 mM, or about 100mM. The pH of the buffer solution comprising the buffer substance isfrom about 2.8 to about 3.2. In particular aspects, the pH is about 3.0.

In general, the pre-equilibration solution will comprise a buffersolution; the equilibration solution will comprise a buffer solution anda water-miscible organic modifier at a concentration of about 4 to 7percent per volume.

Loading the column, chromatography, and elution of the insulin orinsulin analog is achieved using known, conventional technical methods.The loading of the column with a loading solution comprising the insulinor insulin analog to be purified may have a protein content of about 3.0to 48 grams of insulin or insulin analog per liter of reverse phasematerial. In general, the loading of the reverse phase material may beachieved by diluting the eluent from the cation exchange chromatographycomprising the insulin or insulin analog mixture with about 2.8 to 5.2volumes, or about 4 volumes of water to provide a sample solution.

Following the loading of the insulin or insulin analog onto the column,the column is washed in with a first wash solution comprising a buffercomprising about 4 to 7 percent per volume of a water miscible organicmodifier for at least three column volumes. The column wash is followedwith a second wash. The second wash is applied as a liner gradient fromabout 4 to 7 percent to 13 to 15 percent per volume of a water miscibleorganic modifier in a buffer. Elution of the insulin or insulin analogis achieved by applying to the column an elution solution comprising aliner gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer. Elution of theinsulin or insulin analog is detected by UV monitoring. Yield may bemonitored by measuring UV absorbance of fractions collected during theelution and purity may be determined by HPLC.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). The temperature duringthe chromatography and elution is greater than room temperature or 30°C. or greater than 40° C. In particular embodiments, the temperatureduring the chromatography and elution may be about 25° C. to about 50°C. or from about 37° C. to about 47° C. In particular embodiments, thetemperature during the chromatography and elution is greater thangreater than 40° C. In particular aspects, the temperature during thechromatography and elution is about 43° C. or more or about 43° C. toabout 47° C. In particular aspects, the temperature during thechromatography and elution is about 43° C. or at least 43° C.Preferably, the temperature is kept constant or nearly constant over thecourse of the chromatography and elution. The operating pressure duringthe chromatography and elution is substantially constant. Thechromatography and elution may be carried out using a columndifferential pressure that is 5.5 MPa or less.

Concentrating the insulin or insulin analog obtained from the reversephase chromatography may be achieved by precipitation with zinc salt orby crystallization using methods known in the art. The resulting insulinprecipitates may be isolated by decantation, centrifugation, orfiltration, and then dried. The present invention is suitable not onlyfor analytical chromatography but also for preparative chromatography,in particular when the process according to the invention is carried outwith a preparative high-pressure liquid chromatography (HPLC) system.Thus, the present invention may be used in a process for preparinginsulin or insulin analog for use in treating diabetes.

Insulin Analogs

The insulin analog may be an acid-stable insulin analog, which is stableand soluble in acidic or weakly acidic solutions and insoluble orpartially insoluble at physiological pH, or a pI-shifted insulin analogin which the pI of the insulin analog is less than or greater than thepI of native human insulin. The pI of native insulin is 5.4 to 5.6 thusa pI shifted insulin analog has a pI greater than 5.6 or less than 5.4.In particular aspects the insulin analog has a pI from between 5.8 to8.0. An acid-stable insulin analog such as insulin glargine has a pI ofabout 6.7 to 7.0. In a further aspect, the insulin analog is insulinglargine, insulin aspart, insulin glulisine, or insulin lispro. Forchromatography of acid-stable insulins a weak cation material may beused and for chromatography for insulins with a pI similar to that ofnative insulin, a strong cation exchange material may be used.

Thus, in particular embodiments the present invention provides a processfor isolating properly folded acid-stable insulin analog from an aqueousmixture comprising the acid-stable insulin analog and relatedimpurities, wherein the process comprises (a) performing an acid-stablecation exchange chromatography with the aqueous mixture in the presenceof a first water miscible organic modifier and at an elevatedtemperature to yield a first acid-stable insulin analog mixture; and (b)performing a reverse phase high performance liquid chromatography on thefirst acid-stable insulin analog mixture in the presence of a secondwater miscible organic modifier and at an elevated temperature toprovide a second mixture comprising the isolated properly foldedacid-stable insulin analog.

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the second mixture is obtainedfrom the reverse phase high performance liquid chromatography using alinear gradient comprising the second water miscible organic modifierincreasing in concentration from about 13 to 15 percent by volume toabout 25 to 27 percent by volume. In a further aspect of the process,the linear gradient is performed on a column over a course of about 6column volumes and at about a 10 minute residence time. In a furtheraspect of the process, the process is performed on a column and thelinear gradient is performed at a rate of change of about 1.7 percent to2.3 percent of water miscible organic modifier per column volume(Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 2.0 percent of water miscible organic modifier percolumn volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV).

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa. Inparticular aspects of the process, the acid-stable cation exchangechromatography has an outlet temperature greater than room temperature;greater than 30° C.; greater than 40° C.; or a temperature between 41°C. to 45° C.; or about 42° C.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length. Inparticular aspects of the process, the reverse phase high performanceliquid chromatography has an outlet temperature greater than roomtemperature; greater than 30° C.; greater than 40° C.; or a temperaturebetween 40° C. to 46° C.; or about 43° C.

The present invention further provides a process for purifying aproperly folded acid-stable insulin analog from a mixture comprising theacid-stable insulin analog and related impurities, the processcomprising: (a) applying the mixture to a cation exchange chromatographymatrix; (b) washing the matrix with a first wash solution comprising aconcentration of mineral salt of about 10 to 25 mM and a first watermiscible organic modifier and then washing the column with a second washsolution comprising a concentration of mineral salt greater than theconcentration of mineral salt in the first wash solution and less thanthe concentration of mineral salt capable of eluting the acid-stableinsulin analog from the column and the first water miscible organicmodifier; (c) eluting the acid-stable insulin analog from the columnwith an eluting solution comprising the concentration of mineral saltcapable of eluting the acid-stable insulin analog from the column andthe first water miscible organic modifier to provide a second mixture;and (d) applying the second mixture to a reverse phase high performanceliquid chromatography matrix and eluting the acid-stable insulin analogwith a linear gradient of a second water miscible organic modifier ofincreasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedacid-stable insulin analog; wherein the acid-stable cation exchangechromatography is performed at an outlet temperature greater than roomtemperature; greater than 30° C.; greater than 40° C.; or a temperaturebetween 41° C. to 45° C.; or about 42° C. and the reverse phase highperformance liquid chromatography is performed at an outlet temperaturegreater than room temperature; greater than 30° C.; greater than 40° C.;or a temperature between 40° C. to 46° C.; or about 43° C.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step (d): applying the secondmixture to a reverse phase high performance liquid chromatographymatrix; washing the matrix with a first solution comprising about 4 to 7percent of a second water miscible organic modifier; washing the matrixwith a linear gradient of the second water miscible organic modifier ofincreasing concentration from about 4 to 7 percent by volume to about 13to 15 percent by volume; and eluting the insulin or insulin analog witha linear gradient of a second water miscible organic modifier ofincreasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedacid-stable insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl. In particular aspects, the column is washed withabout 5 to 20 volumes of the wash solution.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl. In a further aspect, the column iswashed with about 15 to 25 volumes of the second wash solution.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl. In particular aspects, the acid-stable insulinanalog is eluted from the column with about 20 column volumes of theeluting solution.

The present invention further provides a process for purifying aproperly folded acid-stable insulin analog from a mixture comprising theacid-stable insulin analog and related impurities, the processcomprising:

-   -   (a) a cation exchange chromatography step performed in a column        under a column differential pressure of less than 1.1 MPa, or        less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa and        at an outlet temperature greater than room temperature; greater        than 30° C.; greater than 40° C.; or a temperature between        41° C. to 45° C.; or about 42° C., the steps comprising        -   (i) applying the mixture to a cation exchange chromatography            matrix in the column;        -   (ii) washing the matrix with a first wash solution            comprising a concentration of mineral salt of about 10 to 25            mM and a first water miscible organic modifier and then            washing the column with a second wash solution comprising a            concentration of mineral salt greater than the concentration            of mineral salt in the first wash solution and less than the            concentration of mineral salt capable of eluting the            acid-stable insulin analog from the column and the first            water miscible organic modifier;        -   (iii) eluting the acid-stable insulin analog from the matrix            with an eluting solution comprising the concentration of            mineral salt capable of eluting the acid-stable insulin            analog from the column and the first water miscible organic            modifier to provide a second mixture; and    -   (b) a reverse phase high performance liquid chromatography being        performed at an outlet temperature greater than room        temperature; greater than 30° C.; greater than 40° C.; or a        temperature between 40° C. to 46° C.; or about 43° C., the steps        comprising        -   (i) applying the second mixture to a reverse phase high            performance liquid chromatography matrix and        -   (ii) eluting the insulin or insulin analog with a linear            gradient of a second water miscible organic modifier of            increasing concentration from about 13 to 15 percent by            volume to about 25 to 27 percent by volume to provide a            mixture of the properly folded acid-stable insulin analog.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step (b) a reverse phase highperformance liquid chromatography being performed at an outlettemperature greater than room temperature; greater than 30° C.; greaterthan 40° C.; or a temperature between 40° C. to 46° C.; or about 43° C.,the steps comprising

-   -   (i) applying the second mixture to a reverse phase high        performance liquid chromatography matrix;    -   (ii) washing the matrix with a first solution comprising about 4        to 7 percent of a second water miscible organic modifier;    -   (iii) washing the matrix with a linear gradient of the second        water miscible organic modifier of increasing concentration from        about 4 to 7 percent by volume to about 13 to 15 percent by        volume; and    -   (iv) eluting the insulin or insulin analog with a linear        gradient of a second water miscible organic modifier of        increasing concentration from about 13 to 15 percent by volume        to about 25 to 27 percent by volume to provide a mixture of the        properly folded acid-stable insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl. In particular aspects, the column is washed withabout 5 to 20 volumes of the wash solution.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl. In a further aspect, the column iswashed with about 15 to 25 volumes of the second wash solution.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl. In particular aspects, the acid-stable insulinanalog is eluted from the column with about 20 column volumes of theeluting solution.

In a further aspect of the process, the mixture that is loaded onto thecation exchange chromatography matrix comprises from about 3.0 to about26.0 g of protein per liter in a solution comprising a water miscibleorganic modifier. In a further aspect, the mixture comprises from 10 toabout 50 percent by volume of a water miscible organic modifier. In afurther aspect, the loading step is performed using an aqueous mixturewhich comprises from about 25 to about 35 percent or 30 percent byvolume of a water miscible organic modifier. In particular aspects, thewater miscible organic modifier is hexylene glycol.

In a further aspect of the process, the elution of the acid-stableinsulin analog is isocratic.

The present invention further provides a process for isolating properlyfolded pI-shifted insulin analog from an aqueous mixture comprising thepI-shifted insulin analog and related impurities, wherein the processcomprises (a) performing an acid-stable cation exchange chromatographywith the aqueous mixture in the presence of a first water miscibleorganic modifier and at an elevated temperature to yield a firstpI-shifted insulin analog mixture; and (b) performing a reverse phasehigh performance liquid chromatography on the first pI-shifted insulinanalog mixture in the presence of a second water miscible organicmodifier and at an elevated temperature to provide a second mixturecomprising the isolated properly folded pI-shifted insulin analog.

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the second mixture is obtainedfrom the reverse phase high performance liquid chromatography using alinear gradient comprising the second water miscible organic modifierincreasing in concentration from about 13 to 15 percent by volume toabout 25 to 27 percent by volume. In a further aspect of the process,the linear gradient is performed on a column over a course of about 6column volumes and at about a 10 minute residence time. In a furtheraspect of the process, the process is performed on a column and thelinear gradient is performed at a rate of change of about 1.7 percent to2.3 percent of water miscible organic modifier per column volume(Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 2.0 percent of water miscible organic modifier percolumn volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV).

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa. Inparticular aspects of the process, the acid-stable cation exchangechromatography has an outlet temperature greater than room temperature;greater than 30° C.; greater than 40° C.; or a temperature between 41°C. to 45° C.; or about 42° C.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length. Inparticular aspects of the process, the reverse phase high performanceliquid chromatography has an outlet temperature greater than roomtemperature; greater than 30° C.; greater than 40° C.; or a temperaturebetween 40° C. to 46° C.; or about 43° C.

The present invention further provides a process for purifying aproperly folded pI-shifted insulin analog from a mixture comprising theacid-stable insulin analog and related impurities, the processcomprising: (a) applying the mixture to a cation exchange chromatographymatrix; (b) washing the matrix with a first wash solution comprising aconcentration of mineral salt of about 10 to 25 mM and a first watermiscible organic modifier and then washing the column with a second washsolution comprising a concentration of mineral salt greater than theconcentration of mineral salt in the first wash solution and less thanthe concentration of mineral salt capable of eluting the acid-stableinsulin analog from the column and the first water miscible organicmodifier; (c) eluting the acid-stable insulin analog from the columnwith an eluting solution comprising the concentration of mineral saltcapable of eluting the acid-stable insulin analog from the column andthe first water miscible organic modifier to provide a second mixture;and (d) applying the second mixture to a reverse phase high performanceliquid chromatography matrix and eluting the acid-stable insulin analogwith a linear gradient of a second water miscible organic modifier ofincreasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedacid-stable insulin analog; wherein the acid-stable cation exchangechromatography is performed at an outlet temperature greater than roomtemperature; greater than 30° C.; greater than 40° C.; or a temperaturebetween 41° C. to 45° C.; or about 42° C. and the reverse phase highperformance liquid chromatography is performed at an outlet temperaturegreater than room temperature; greater than 30° C.; greater than 40° C.;or a temperature between 40° C. to 46° C.; or about 43° C.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step (d): applying the secondmixture to a reverse phase high performance liquid chromatographymatrix; washing the matrix with a first solution comprising about 4 to 7percent of a second water miscible organic modifier; washing the matrixwith a linear gradient of the second water miscible organic modifier ofincreasing concentration from about 4 to 7 percent by volume to about 13to 15 percent by volume; and eluting the insulin or insulin analog witha linear gradient of a second water miscible organic modifier ofincreasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedacid-stable insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl. In particular aspects, the column is washed withabout 5 to 20 volumes of the wash solution.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl. In a further aspect, the column iswashed with about 15 to 25 volumes of the second wash solution.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl. In particular aspects, the acid-stable insulinanalog is eluted from the column with about 20 column volumes of theeluting solution.

The present invention further provides a process for purifying aproperly folded pI-shifted insulin analog from a mixture comprising theacid-stable insulin analog and related impurities, the processcomprising:

-   -   (a) a cation exchange chromatography step performed in a column        under a column differential pressure of less than 1.1 MPa, or        less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa and        at an outlet temperature greater than room temperature; greater        than 30° C.; greater than 40° C.; or a temperature between        41° C. to 45° C.; or about 42° C., the steps comprising        -   (i) applying the mixture to a cation exchange chromatography            matrix in the column;        -   (ii) washing the matrix with a first wash solution            comprising a concentration of mineral salt of about 10 to 25            mM and a first water miscible organic modifier and then            washing the column with a second wash solution comprising a            concentration of mineral salt greater than the concentration            of mineral salt in the first wash solution and less than the            concentration of mineral salt capable of eluting the            acid-stable insulin analog from the column and the first            water miscible organic modifier;        -   (iii) eluting the acid-stable insulin analog from the matrix            with an eluting solution comprising the concentration of            mineral salt capable of eluting the acid-stable insulin            analog from the column and the first water miscible organic            modifier to provide a second mixture; and    -   (b) a reverse phase high performance liquid chromatography being        performed at an outlet temperature greater than room        temperature; greater than 30° C.; greater than 40° C.; or a        temperature between 40° C. to 46° C.; or about 43° C., the steps        comprising        -   (i) applying the second mixture to a reverse phase high            performance liquid chromatography matrix and        -   (ii) eluting the insulin or insulin analog with a linear            gradient of a second water miscible organic modifier of            increasing concentration from about 13 to 15 percent by            volume to about 25 to 27 percent by volume to provide a            mixture of the properly folded acid-stable insulin analog.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step (b) a reverse phase highperformance liquid chromatography being performed at an outlettemperature greater than room temperature; greater than 30° C.; greaterthan 40° C.; or a temperature between 40° C. to 46° C.; or about 43° C.,the steps comprising

-   -   (i) applying the second mixture to a reverse phase high        performance liquid chromatography matrix;    -   (ii) washing the matrix with a first solution comprising about 4        to 7 percent of a second water miscible organic modifier;    -   (iii) washing the matrix with a linear gradient of the second        water miscible organic modifier of increasing concentration from        about 4 to 7 percent by volume to about 13 to 15 percent by        volume; and    -   (iv) eluting the insulin or insulin analog with a linear        gradient of a second water miscible organic modifier of        increasing concentration from about 13 to 15 percent by volume        to about 25 to 27 percent by volume to provide a mixture of the        properly folded acid-stable insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl. In particular aspects, the column is washed withabout 5 to 20 volumes of the wash solution.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl. In a further aspect, the column iswashed with about 15 to 25 volumes of the second wash solution.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl. In particular aspects, the acid-stable insulinanalog is eluted from the column with about 20 column volumes of theeluting solution.

In a further aspect of the process, the mixture that is loaded onto thecation exchange chromatography matrix comprises from about 3.0 to about26.0 g of protein per liter in a solution comprising a water miscibleorganic modifier. In a further aspect, the mixture comprises from 10 toabout 50 percent by volume of a water miscible organic modifier. In afurther aspect, the loading step is performed using an aqueous mixturewhich comprises from about 25 to about 35 percent or 30 percent byvolume of a water miscible organic modifier. In particular aspects, thewater miscible organic modifier is hexylene glycol.

In a further aspect of the process, the elution of the pI-shiftedinsulin analog is isocratic.

The present invention further provides a process for isolating properlyfolded insulin glargine from an aqueous mixture comprising the insulinglargine and related impurities, wherein the process comprises (a)performing an acid-stable cation exchange chromatography with theaqueous mixture in the presence of a first water miscible organicmodifier and at an elevated temperature to yield a first insulinglargine mixture; and (b) performing a reverse phase high performanceliquid chromatography on the first insulin glargine mixture in thepresence of a second water miscible organic modifier and at an elevatedtemperature to provide a second mixture comprising the isolated properlyfolded insulin glargine.

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the second mixture is obtainedfrom the reverse phase high performance liquid chromatography using alinear gradient comprising the second water miscible organic modifierincreasing in concentration from about 13 to 15 percent by volume toabout 25 to 27 percent by volume. In a further aspect of the process,the linear gradient is performed on a column over a course of about 6column volumes and at about a 10 minute residence time. In a furtheraspect of the process, the process is performed on a column and thelinear gradient is performed at a rate of change of about 1.7 percent to2.3 percent of water miscible organic modifier per column volume(Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 2.0 percent of water miscible organic modifier percolumn volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV).

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa. Inparticular aspects of the process, the acid-stable cation exchangechromatography has an outlet temperature greater than room temperature;greater than 30° C.; greater than 40° C.; or a temperature between 41°C. to 45° C.; or about 42° C.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length. Inparticular aspects of the process, the reverse phase high performanceliquid chromatography has an outlet temperature greater than roomtemperature; greater than 30° C.; greater than 40° C.; or a temperaturebetween 40° C. to 46° C.; or about 43° C.

The present invention further provides a process for purifying aproperly folded insulin glargine from a mixture comprising theacid-stable insulin analog and related impurities, the processcomprising: (a) applying the mixture to a cation exchange chromatographymatrix; (b) washing the matrix with a first wash solution comprising aconcentration of mineral salt of about 10 to 25 mM and a first watermiscible organic modifier and then washing the column with a second washsolution comprising a concentration of mineral salt greater than theconcentration of mineral salt in the first wash solution and less thanthe concentration of mineral salt capable of eluting the acid-stableinsulin analog from the column and the first water miscible organicmodifier; (c) eluting the acid-stable insulin analog from the columnwith an eluting solution comprising the concentration of mineral saltcapable of eluting the acid-stable insulin analog from the column andthe first water miscible organic modifier to provide a second mixture;and (d) applying the second mixture to a reverse phase high performanceliquid chromatography matrix and eluting the acid-stable insulin analogwith a linear gradient of a second water miscible organic modifier ofincreasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedacid-stable insulin analog; wherein the acid-stable cation exchangechromatography is performed at an outlet temperature greater than roomtemperature; greater than 30° C.; greater than 40° C.; or a temperaturebetween 41° C. to 45° C.; or about 42° C. and the reverse phase highperformance liquid chromatography is performed at an outlet temperaturegreater than room temperature; greater than 30° C.; greater than 40° C.;or a temperature between 40° C. to 46° C.; or about 43° C.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the acidic cation exchangechromatography is performed with a differential pressure of less than1.1 MPa, less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step (d): applying the secondmixture to a reverse phase high performance liquid chromatographymatrix; washing the matrix with a first solution comprising about 4 to 7percent of a second water miscible organic modifier; washing the matrixwith a linear gradient of the second water miscible organic modifier ofincreasing concentration from about 4 to 7 percent by volume to about 13to 15 percent by volume; and eluting the insulin or insulin analog witha linear gradient of a second water miscible organic modifier ofincreasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedacid-stable insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl. In particular aspects, the column is washed withabout 5 to 20 volumes of the wash solution.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl. In a further aspect, the column iswashed with about 15 to 25 volumes of the second wash solution.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl. In particular aspects, the acid-stable insulinanalog is eluted from the column with about 20 column volumes of theeluting solution.

The present invention further provides a process for purifying aproperly folded insulin glargine from a mixture comprising theacid-stable insulin analog and related impurities, the processcomprising:

-   -   (a) a cation exchange chromatography step performed in a column        under a column differential pressure of less than 1.1 MPa, or        less than 1.0 MPa, or less than 0.5 MPa, or about 0.276 MPa and        at an outlet temperature greater than room temperature; greater        than 30° C.; greater than 40° C.; or a temperature between        41° C. to 45° C.; or about 42° C., the steps comprising        -   (i) applying the mixture to a cation exchange chromatography            matrix in the column;        -   (ii) washing the matrix with a first wash solution            comprising a concentration of mineral salt of about 10 to 25            mM and a first water miscible organic modifier and then            washing the column with a second wash solution comprising a            concentration of mineral salt greater than the concentration            of mineral salt in the first wash solution and less than the            concentration of mineral salt capable of eluting the            acid-stable insulin analog from the column and the first            water miscible organic modifier;        -   (iii) eluting the acid-stable insulin analog from the matrix            with an eluting solution comprising the concentration of            mineral salt capable of eluting the acid-stable insulin            analog from the column and the first water miscible organic            modifier to provide a second mixture; and    -   (b) a reverse phase high performance liquid chromatography being        performed at an outlet temperature greater than room        temperature; greater than 30° C.; greater than 40° C.; or a        temperature between 40° C. to 46° C.; or about 43° C., the steps        comprising        -   (i) applying the second mixture to a reverse phase high            performance liquid chromatography matrix and        -   (ii) eluting the insulin or insulin analog with a linear            gradient of a second water miscible organic modifier of            increasing concentration from about 13 to 15 percent by            volume to about 25 to 27 percent by volume to provide a            mixture of the properly folded acid-stable insulin analog.

In a further aspect of the process, the linear gradient is performed ona column over a course of about 6 column volumes and at about a 10minute residence time. In a further aspect of the process, the processis performed on a column and the linear gradient is performed at a rateof change of about 1.7 percent to 2.3 percent of water miscible organicmodifier per column volume (Δ1.7%/CV to Δ2.3%/CV). In a further aspectof the process, the process is performed on a column and the lineargradient is performed at a rate of change of about 2.0 percent of watermiscible organic modifier per column volume (Δ2.0%/CV). In a furtheraspect of the process, the process is performed on a column and thelinear gradient is from 11 percent to 27 percent by volume of watermiscible organic modifier or performed at a rate of change of about 2.6percent of water miscible organic modifier per column volume (Δ2.0%/CV).

In particular aspects of the process, the first water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the first water miscible organic modifier is hexylene glycol.The first water miscible organic modifier may be at a concentrationbetween 1 to 50 percent by volume or 20 to 50 percent by volume. In afurther aspect, the first water miscible organic modifier may be at aconcentration between 25 to 40 percent by volume. In a further aspect,the first water miscible organic modifier may be at a concentrationbetween 5 to 30 percent by volume. In particular aspects, the watermiscible organic modifier is at a concentration of about 30 percent byvolume.

In particular aspects of the process, the second water miscible organicmodifier is selected from acetonitrile, ketones, ethyl acetate, alcoholshaving 1 to 4 carbon atoms, and polyols having 3 to 6 carbon atoms. Inparticular aspects, the alcohol is selected from 1 or 2-propanol (n oriso-propanol), methanol, ethanol, and hexylene glycol. In a furtheraspect, the second water miscible organic modifier is isopropanol.

In particular aspects of the process, the acidic cation exchangematerial is a weak cation exchanger comprising carboxy or carboxymethylfunctional groups. In particular aspects, the acidic cation exchangematerial is a strong cation exchanger comprising sulfonic acidfunctional groups. In a further aspect of the process, the acidic cationexchange material is temperature-stable, which in particular aspects maycomprise a high-capacity hydrogel polymerized within the pores of arigid ceramic bead and cross-linked to sulfo groups on the bead. Thus,the temperature-stable acidic cation exchange material comprises ahigh-capacity hydrogel polymerized within the pores of rigid ceramicbeads and cross-linked to sulfo groups on the beads. In particularaspects, the hydrogel comprises carboxy or carboxymethyl functionalgroups, or sulfonic acid functional groups.

In particular aspects of the process, the reverse phase high performanceliquid chromatography is performed with a temperature stable andpressure stable organic modified chromatography material. In particularaspects, the chromatography material is a silica-based reverse phaseresin modified with hydrocarbon chains 4 to 18 carbons in length. In afurther aspect, the hydrocarbon chains are about 8 carbons in length.

In a further aspect of the process, in step (b) a reverse phase highperformance liquid chromatography being performed at an outlettemperature greater than room temperature; greater than 30° C.; greaterthan 40° C.; or a temperature between 40° C. to 46° C.; or about 43° C.,the steps comprising

-   -   (i) applying the second mixture to a reverse phase high        performance liquid chromatography matrix;    -   (ii) washing the matrix with a first solution comprising about 4        to 7 percent of a second water miscible organic modifier;    -   (iii) washing the matrix with a linear gradient of the second        water miscible organic modifier of increasing concentration from        about 4 to 7 percent by volume to about 13 to 15 percent by        volume; and    -   (iv) eluting the insulin or insulin analog with a linear        gradient of a second water miscible organic modifier of        increasing concentration from about 13 to 15 percent by volume        to about 25 to 27 percent by volume to provide a mixture of the        properly folded acid-stable insulin analog.

In particular aspects of the process, the second wash is achieved usinga linear gradient from about 4 to 7 percent to 13 to 15 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 0.5 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 12 percentto 22 percent of water miscible organic modifier per column volume(Δ12%/CV to Δ22%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is performed at a rate ofchange of about 18 percent of water miscible organic modifier per columnvolume (Δ18%/CV). In a further aspect of the process, the process isperformed on a column and the linear gradient is from 5 percent to 11percent by volume of water miscible organic modifier or performed at arate of change of about 12 percent of water miscible organic modifierper column volume (Δ12%/CV).

In particular aspects of the process, the elution is achieved using alinear gradient from about 13 to 15 percent to 25 to 27 percent pervolume of a water miscible organic modifier in a buffer over a course ofabout 6 column volumes and at about a 10 minute residence time. In afurther aspect of the process, the process is performed on a column andthe linear gradient is performed at a rate of change of about 1.7percent to 2.3 percent of water miscible organic modifier per columnvolume (Δ1.7%/CV to Δ2.3%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is performed ata rate of change of about 2.0 percent of water miscible organic modifierper column volume (Δ2.0%/CV). In a further aspect of the process, theprocess is performed on a column and the linear gradient is from 11percent to 27 percent by volume of water miscible organic modifier orperformed at a rate of change of about 2.6 percent of water miscibleorganic modifier per column volume (Δ2.6%/CV). In a further aspect ofthe process, the cation exchange chromatography first wash solutioncomprises a concentration of mineral salt of about 10 mM to about 20 mM,which in particular aspects may be NaCl. In a further aspect of theprocess, the first wash solution comprises a concentration of mineralsalt of about 18 mM to about 22 mM, which in particular aspects may beNaCl. In a particular aspect, the first wash solution comprises aconcentration of mineral salt of about 20 mM, which in particularaspects may be NaCl. In particular aspects, the column is washed withabout 5 to 20 volumes of the wash solution.

In a further aspect, the cation exchange chromatography second washsolution comprises a concentration of mineral salt of about 35 mM to 39mM of a mineral salt, which in particular aspects may be NaCl. In afurther aspect, the concentration of mineral salt is about 37 mM, whichin particular aspects may be NaCl. In a further aspect, the column iswashed with about 15 to 25 volumes of the second wash solution.

In further aspects of the process, the cation exchange chromatographyeluting solution comprises a concentration of mineral salt of about 80to 100 mM, which in particular aspects may be NaCl. In further aspectsof the process, the eluting solution comprises a concentration ofmineral salt of about 93 to 97 mM, which in particular aspects may beNaCl. In further aspects of the process, the eluting solution comprisesa concentration of mineral salt of about 95 mM, which in particularaspects may be NaCl. In particular aspects, the acid-stable insulinanalog is eluted from the column with about 20 column volumes of theeluting solution.

In a further aspect of the process, the mixture that is loaded onto thecation exchange chromatography matrix comprises from about 3.0 to about26.0 g of protein per liter in a solution comprising a water miscibleorganic modifier. In a further aspect, the mixture comprises from 10 toabout 50 percent by volume of a water miscible organic modifier. In afurther aspect, the loading step is performed using an aqueous mixturewhich comprises from about 25 to about 35 percent or 30 percent byvolume of a water miscible organic modifier. In particular aspects, thewater miscible organic modifier is hexylene glycol.

In a further aspect of the process, the elution of the insulin glargineis isocratic.

The following examples are intended to promote a further understandingof the present invention.

EXAMPLE 1

The acidic cation exchange (CEX) column is packed with BioSepra CMCeramic HyperD F, a high-capacity hydrogel polymerized within the poresof a rigid ceramic bead available from Pall Corporation, PortWashington, N.Y.; Cat. No. 20050. The defined volume of cation exchangematerial is first allowed to settle. The storage solution (20% ethanol)is decanted and replaced with an equal volume of CEX Strip/StorageSolution (Table 1) and the cation exchange material is resuspended inthis solution. Settling, decanting, and resuspension with an equalvolume of CEX Strip/Storage Solution are repeated two more timesallowing for the sufficient equilibration of the cation exchangematerial with 30% hexylene glycol.

Next, the slurry is diluted to achieve a slurry percent of about 50% andheld in the CEX Strip/Storage Solution for 24 hours. The cation exchangematerial slurry is transferred to the column and resuspended with apaddle. The upper head plate is inserted into the column and lowered tothe solution interface. The column is flow-packed with CEX Strip/StorageSolution at a flow rate that produces a column differential pressure ofabout 40 psid (0.276 MPA) or more and at a temperature of about 20±2° C.Once a stable bed height is achieved, the flow rate is reduced and theupper head plate is lowered until about 2 to 3 mm above the cationexchange material interface. The head space is to prevent the cationexchange material from interfering with the sealing of the head plate.The column is returned to the packing pressure, and the bed is allowedto compress again. The procedure of slowing flow and lowering the headplate is repeated as necessary until the upper head plate is about 2 to3 mm above the resin cation exchange material interface at the packingpressure.

Next, the column is connected to the chromatography skid and flushedwith CEX Cleaning Solution (Table 1) as a pre-use sanitization, duringwhich a final compression of the bed is observed. If necessary, the headplate should be lowered again to remain 2 to 3 mm above the cationexchange material interface.

Next, the column is promptly flushed with CEX Pre-Equilibration Solution(Table 1) and CEX Strip/Storage Solution.

TABLE 1 CEX Chromatography Buffers & Raw Materials Buffers/Raw MaterialDescription CEX Chromatography cation BioSepra CM Ceramic HyperD Fexchange material CEX Load Diluent Hexylene Glycol CEX Pre-Equilibration1M sodium acetate, pH 5.1 Solution CEX Equilibration/Wash 1 20 mM sodiumacetate, 20 mM sodium chloride, 30% hexylene Solution (A1) glycol (v/v),pH 5.1 (before hexylene glycol addition) CEX Strip/Storage Solution 20mM sodium acetate, 250 mM sodium chloride, 30% hexylene (B1) glycol(v/v), pH 5.1 (before hexylene glycol addition) CEX Cleaning Solution0.5N sodium hydroxide CEX Load Filter Sartopore 2 (0.45/0.2 μm) MaxiCapSize 1 (0.6 m²)

Next, the column is equilibrated with CEX Equilibration/Wash 1 Solution(Table 1) using a gradient initially to transition from the CEXStrip/Storage Solution to the CEX Equilibration/Wash 1 Solution atcolumn differential pressure of about 40 psid (0.276 MPa) or less withan outlet temperature of about 40±2° C. Next, the column is flushed withthe defined volume of CEX Strip/Storage Solution and the (low-to-high)transition is monitored via conductivity. Finally, the column may bestored until use.

Crude recombinant insulin or insulin analog heterodimer obtained from atrypsin digest in 50 mM acetic acid, pH 3.5 is diluted to about 0.3 to0.4 g/L with hexylene glycol prior to loading on the column and the pHadjusted to about 4.2. The mass of hexylene glycol to crude recombinantinsulin is about 0.395 kg hexylene glycol/kg crude recombinant insulin.To prepare for loading of the diluted crude recombinant insulin, thecolumn is equilibrated with CEX Equilibration/Wash 1 Solution using agradient initially to transition from the CEX Strip/Storage Solution tothe CEX Equilibration/Wash 1 Solution to prevent extreme pH excursionsacross the cation exchange material in the column. The column is loadedwith the diluted crude recombinant insulin or insulin analog at about3-6 g insulin or insulin analog/L cation exchange material/cycle. Allsteps between and including column wash and column strip use CEXEquilibration/Wash 1 Solution (A1) and CEX Strip/Storage Solution (B1)(Table 1). Following loading, the column is washed with CEXEquilibration/Wash 1 Solution containing about 20 mM NaCl.

The column is operated a flow rate that produces a column differentialpressure of about 40 psid (0.276 MPa) or less with an outlet temperatureof about 42±2° C. In general, the flow rate is about 182 to 201 cm/houror about 1 L/minute. The column is washed as shown in FIG. 1 with anA1/B1 mixture containing about 6.5±1.0% solution B1 in solution A1 toproduce a solution comprising about 37 mM NaCl for about 18-20 columnvolumes. The column is then eluted as shown in FIG. 1 with an A1/B1mixture containing about 26.1±1.0% B1 in A1 to produce a solutioncomprising about 95 mM NaCl with about 20 column volumes. Duringelution, fractions are collected during the elution process to allowanalysis by UV absorption prior to pooling of those fractions thatcontain eluted insulin or insulin analog. In general, collection offractions begins at the start of the elution step and ends when the UVpeak is below 10% of the peak maximum value. The fractions may beanalyzed by HPLC to determine purity. The fractions that correspond tothe required purity are combined. In general, purity as measured by HPLCis greater than 90% with a yield of about 60-85% or more.

A reverse phase (RP) high performance liquid chromatography column ispacked with KROMOSIL. RP Chromatography materials are shown in Table 2.The column is then equilibrated at 43° C. using 5% Buffer B (5%isopropanol and 95% Buffer A). The column outlet temperature iscontrolled by an inline heat exchanger and column jacket. The column isstored in a solution of 30% Buffer A and 70% Buffer B prepared inlineusing the Step Operation: For processing, the RP load is prepared bydiluting (inline) the CEX product pool 4× with water. During theprocess, UV absorbance is monitored at 295 nm.

The column is then loaded with diluted CEX pool to a defined load factorof about less than 24 g/L of protein. The column is then washed withWash 1 followed by Wash 2. Wash 2 is a linear gradient of 5 to 14percent isopropanol in Buffer A. The insulin or insulin analog productis then eluted with a linear gradient of 14 to 26% isopropanol in BufferA. The product is collected based on A295 values into chilled water toreduce the isopropanol concentration in the product to less than 10%.Following elution, the column is stripped and re-equilibrated. Thechromatography gradient used during the RP method from wash throughre-equilibration is shown in FIG. 2.

TABLE 2 RP Chromatography Buffers & Raw Materials Buffers/Raw MaterialDescription RP Chromatography Akzo Nobel KROMASIL 100-10-C8 resinmaterial (100 Å pore size, 10 μm particle size, C8 bonded phase) BufferA 100 mM ammonium acetate, pH 3.0 Buffer B HPLC grade isopropanol(2-propanol, IPA) Wash 1 95% Buffer A 5% Buffer B Wash 2 Start gradient:95% Buffer A, 5% Buffer B End gradient: 86% Buffer A, 14% Buffer BElution buffer Start gradient: 86% Buffer A, 14% Buffer B End gradient:74% Buffer A, 26% Buffer B

EXAMPLE 2

A crude mixture of the acid-stable insulin analog Gly(A21), Arg(B31),Arg(B32)-human insulin (insulin glargine) is purified as in Example 1.Because of non-specific enzymatic cleavages during the trypsin digest,very small amounts of product-related impurities, e.g. the three aminoacid B-chain truncate (des-Thr) are produced. These product impurities,which are present in the crude mixture, are reduced using the method inExample 1. In general, the yield of the process has been about 65% witha purity of greater than 99.0% as determined by HPLC analysis.

Table of Sequences SEQ ID NO: Description Sequence 1Human insulin A chain GIVEQCCTSICSLYQLENYCN 2 Human insulin B chainFVNQHLCGSHLVEALYLVCGERGFFYTPKT 3 Insulin glargine A chainGIVEQCCTSICSLYQLENYCG 4 Insulin glargine B chainFVNQHLCGSHLVEALYLVCGERGFFYTPKTRR 5 Insulin lispro B chainFVNQHLCGSHLVEALYLVCGERGFFYTKPT 6 Insulin glusiline B chainFVKQHLCGSHLVEALYLVCGERGFFYTPET

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

What is claimed is:
 1. A process for isolating properly folded insulinor insulin analog from an aqueous mixture comprising the insulin orinsulin analog and related impurities, wherein the process comprises:(a) performing an acid-stable cation exchange chromatography with theaqueous mixture at a differential pressure of about 0.276 MPa in thepresence of hexylene glycol at a concentration of about 30% by volumeand at an outlet temperature between 41° C. to 45° C. under isocraticconditions to yield a first insulin or insulin analog mixture; and (b)performing a reverse phase high performance liquid chromatography with asilica-based reverse phase resin on the first insulin or insulin analogmixture in the presence of isopropanol and at an outlet temperaturebetween 40° C. to 46° C. to provide a second mixture comprising theisolated properly folded insulin or insulin analog.
 2. The process ofclaim 1, wherein the acid-stable cation exchange chromatography has anoutlet temperature of about 42° C.
 3. The process of claim 1, whereinthe reverse phase high performance liquid chromatography has an outlettemperature of about 43° C.
 4. The process of claim 1, wherein theinsulin analog comprises an acid-stable insulin analog, which is stableand soluble in acidic or weakly acidic solutions and insoluble orpartially insoluble at physiological pH.
 5. The process of claim 4,wherein the acid-stable insulin analog comprises insulin glargine. 6.The process of claim 1, wherein the insulin analog comprises apI-shifted insulin analog in which the pI of the insulin analog is lessthan or greater than the pI of native human insulin.
 7. The process ofclaim 6, wherein the pI of the pI shifted insulin analog comprises a pIgreater than 5.6 or less than 5.4.
 8. The process of claim 6, whereinthe pI-shifted insulin analog comprises a pI from between 5.8 to 8.0. 9.The process of claim 1, wherein the insulin analog comprises an insulinanalog selected from the group consisting of insulin glargine, insulinaspart, insulin glulisine, and insulin lispro.
 10. A process forpurifying a properly folded insulin or insulin analog from a crudemixture comprising the insulin or insulin analog and related impurities,the process comprising: (a) providing a crude mixture of insulin orinsulin analog in 50 mM acetic acid, pH 3.5 and diluting the crudemixture with hexylene glycol to about 0.3 to 0.4 g/L and then adjustingthe pH to about 4.2 to produce a diluted crude mixture, wherein the massof hexylene glycol to insulin or insulin analog in the diluted crudemixture is about 0.395 kg hexylene glycol/kg insulin or insulin analog;(b) applying the diluted crude mixture to a temperature-stable cationexchange chromatography matrix; (c) washing the matrix with a first washsolution comprising a concentration of mineral salt of about 10 to 25 mMand hexylene glycol at a concentration of about 30% by volume and thenwashing the matrix with a second wash solution comprising aconcentration of mineral salt greater than the concentration of mineralsalt in the first wash solution and less than the concentration ofmineral salt capable of eluting the insulin or insulin analog from thematrix wherein the second wash solution comprises hexylene glycol at aconcentration of about 30% by volume; (d) eluting the insulin or insulinanalog from the matrix with an eluting solution comprising theconcentration of mineral salt capable of eluting the insulin or insulinanalog from the matrix and hexylene glycol at a concentration of about30% by volume to provide a second mixture; the temperature-stable cationexchange chromatography being performed under a differential pressure ofabout 0.276 MPa and at an outlet temperature of about 42° C.; and (e)diluting the second mixture with water to provide a diluted secondmixture comprising the insulin or insulin analog at a concentration ofless than 24 g/L and applying the diluted second mixture to a reversephase high performance liquid chromatography matrix comprising anacetate buffer at a pH of about 3.0 and isopropanol at a concentrationof about 5% by volume; and eluting the insulin or insulin analog with alinear gradient of isopropanol in an acetate buffer at a pH of about 3.0increasing concentration from about 13 to 15 percent by volume to about25 to 27 percent by volume to provide a mixture of the properly foldedinsulin or insulin analog; the reverse phase high performance liquidchromatography being performed at an outlet temperature of about 43° C.11. The process of claim 10, wherein step (e) further comprises washingthe matrix with a first solution comprising about 4 to 7 percent byvolume of isopropanol in an acetate buffer at a pH of about 3.0; andthen washing the matrix with a linear gradient of the isopropanol in anacetate buffer at a pH of about 3.0 of increasing concentration fromabout 4 to 7 percent by volume to about 13 to 15 percent by volume. 12.The process of claim 10, wherein the insulin analog comprises anacid-stable insulin analog, which is stable and soluble in acidic orweakly acidic solutions and insoluble or partially insoluble atphysiological pH.
 13. The process of claim 12, wherein the acid-stableinsulin analog comprises insulin glargine.
 14. The process of claim 10,wherein the insulin analog comprises a pI-shifted insulin analog inwhich the pI of the insulin analog is less than or greater than the pIof native human insulin.
 15. The process of claim 14, wherein the pI ofthe pI shifted insulin analog comprises a pI greater than 5.6 or lessthan 5.4.
 16. The process of claim 14, wherein the pI-shifted insulinanalog comprises a pI from between 5.8 to 8.0.
 17. The process of claim10, wherein the insulin analog comprises an insulin analog selected fromthe group consisting of insulin glargine, insulin aspart, insulinglulisine, and insulin lispro.